KR101298442B1 - Ophthalmic devices and related methods and compositions - Google Patents

Abstract

Apparatus, methods and compositions are described for improving vision or treating eye diseases, disorders or injuries. Ophthalmic devices, such as corneal onlays, corneal inlays, and penetrating corneal implants, are prepared as materials that are effective in facilitating nerve growth through or on the device. The substance may comprise more than 1% (w / w), for example about 10 to about 30% (w / w) collagen. The material may comprise collagen-polymers and / or second biopolymers or water soluble synthetic polymers crosslinked using EDC / NHS. The material may further comprise a synthetic polymer. The device is positioned within the eye to correct or improve a person's vision or to treat a disease, disorder or injury of the eye of the individual.

Corneal Onlay, Corneal Inlay, Penetrating Corneal Implants, Collagen, Nerve Growth

Description

Ophthalmic device and related methods and compositions {OPHTHALMIC DEVICES AND RELATED METHODS AND COMPOSITIONS}

Cross-reference to related application

This application claims priority based on US Provisional Application No. 60 / 601,270, filed August 13, 2004, which is incorporated by reference in its entirety herein.

1. Field of the Invention

The present invention relates to devices, methods and compositions for improving a person's vision or for treating a traumatic or ophthalmic disease or disorder of an eye of an individual. In particular, the present invention relates to corneal onlays, corneal inlays and corneal implants made with materials that provide one or more advantages to an individual.

2. Description of the field

US Pat. No. 5,713,957 discloses a corneal onlay comprising a non-biodegradable non-hydrogel ocular biodegradable material having pores sufficient to allow onlay penetration of a tissue fluid component having a molecular fluid weight greater than 10,000 Daltons. Is disclosed.

U. S. Patent No. 5,716, 633 discloses collagen / PHEMA-hydrogels that promote the growth of epithelial cells and the regeneration of the stroma. Collagen-hydrogels can be provided as ophthalmic lenses attached to the Bowman's membrane, which are effective in promoting and helping the growth of epithelial cells or adhesion of the corneal epithelium to the front of the lens. Collagen-hydrogels are hydrogel polymers formed by free radical polymerization of a hydrophilic monomer solution gelled and crosslinked in the presence of an aqueous stock solution of collagen to form a three-dimensional polymer net for anchoring collagen. The final concentration of collagen in the onlay is about 0.3 to about 0.5% (w / w).

U. S. Patent No. 5,836, 313 discloses a method of making the implantable composite keratoprosthese. This method provides Keratoprosdes designed to provide a substrate suitable for the growth of corneal epithelial cells. Keratoprosdes are prepared by placing a corneal tissue in a mold having a corneal graft shape and crosslinking the polymer solution to chemically bind the biodegradable hydrogel having a thickness of about 50 to 100 microns to the corneal tissue. Alternatively, the polymer solution is placed between the corneal tissue and the pre-formed hydrogel and polymerized so that the polymer solution is coupled to both the hydrogel and the corneal tissue.

U. S. Patent No. 6,454, 800 discloses a corneal onlay or corneal implant comprising a surface having a plurality of surface indentations that aid in the attachment and growth of tissue cells.

U. S. Patent No. 6,689, 165 discloses a synthetic device for corneal enhancement and replacement using tethered corneal promoters to enhance adhesion and migration of corneal epithelial cells.

Some problems associated with existing collagen-based materials may lead to undesirable light scattering, because collagen-based materials are not optically apparent, perhaps due to the formation or conversion to fiber-based materials. Is the point.

Thus, there is a need for a biodegradable, ophthalmically acceptable material suitable for being placed in the eye for improving the vision of the individual.

Summary of the Invention

The ophthalmic device includes a body comprising a composition effective when facilitating nerve growth through or on the body when located in the eye of an individual. In certain embodiments, the device is a vision enhancing ophthalmic device. In another embodiment, the device is a therapeutic ophthalmic device. It can be understood that the vision enhancing device of the present invention is a device configured to correct one or more refractive errors. In other words, it can be understood that the apparatus of the present invention is a refractive error correcting apparatus. The body of the device of the present invention may be formed to have optical power.

The composition of the present invention is optically clear and may comprise about 1 to about 50% (w / v or w / w) collagen. In certain embodiments, the amount of collagen is greater than 2.5% (w / w or w / v). As used herein, the amount of collagen and / or compositions and other components of the device is to be understood as being w / w or w / v% without departing from the concept of the present invention. In further embodiments, the amount of collagen is greater than about 5.0%. For example, the substance may comprise about 10 to about 30% collagen. In certain embodiments, the material comprises about 1 to about 50% crosslinked collagen, wherein the collagen is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; CAS # 1892-57- 5) and N-hydroxysuccinimide. In further embodiments, the amount of crosslinked collagen is 2.5 to about 50%. The material may comprise a first collagen polymer crosslinked to a second collagen polymer. In certain embodiments, the ophthalmic device disclosed herein is prepared without glutaraldehyde. For example, glutaraldehyde is not used as a crosslinking agent in the manufacturing process of an ophthalmic device. Glutaraldehyde may not be suitable or desirable for use as a crosslinking agent because of handling and safety requirements with respect to glutaraldehyde and / or the compositions and devices of the present invention. In certain embodiments, ophthalmic devices are made without a cytotoxic component, in other words, with components that have reduced cytotoxicity.

The device may be a corneal onlay, corneal inlay or full-thickness corneal implant, such as a device configured to replace the individual's original cornea. The device of the present invention is transparent and can be made from a transparent composition even before it is formed into a device.

The material of the device may comprise one or more cell growth promoters or one or more additional biopolymers.

A method for producing an ophthalmic device, such as a refractive error correcting device, according to the present disclosure, includes 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC and NHS). And crosslinking the collagen polymer. Crosslinking occurs at an acidic pH, for example at a pH of about 5.0 to about 5.5. The method may comprise one or more steps of adding a cellular enterostimulator to the crosslinked composition. The method includes placing the composition into a mold and allowing the composition to cure, forming an ophthalmic device.

Any aspect or combination of aspects described herein, unless the aspects contained in any such combination are inconsistent with each other, as will be apparent from the content and specification herein, and the ordinary skill in the art. It is included in a category. In addition, any aspect or combination of aspects may be specifically excluded from any embodiment of the present invention.

Further advantages and aspects of the present invention will become apparent from the following detailed description, drawings, examples and claims.

1 is a cross-sectional view of a T-adapter of a system for the preparation of the compositions and devices of the present invention.

2 is a cross-sectional view of a female Luer adapter of a system for the manufacture of the compositions and devices of the present invention.

FIG. 3 is a top view of the T-adapter of FIG. 1 with one partition and two syringes coupled thereto for the manufacture of the compositions and devices of the present invention. FIG.

4 is a graph of cell number as a function of time for human recombinant hydrogel material labeled F1.

5 is a graph of cell number as a function of time for human recombinant hydrogel material labeled F3.

6 is a graph of cell number as a function of time for human recombinant hydrogel material labeled F6.

7 is a photograph of human recombinant hydrogel material labeled F3, located in rats.

8 illustrates one embodiment of the device for refractive error correction ophthalmology of the present invention.

8A shows the lens edge shape of one embodiment of an onlay of the present invention.

9 is a graph of expansion ratio as a function of EDC to NH ₂ molar ratio.

10 is a graph of tensile strength as a function of EDC to NH ₂ molar ratio.

11 is a graph of tensile strength as a function of collagen concentration (left) and a graph of expansion ratio as a function of collagen concentration (right).

12 is a graph of heat flow as a function of temperature for compositions with different EDC to NH ₂ molar ratios (left), and a graph of heat flow as a function of temperature for compositions with different CSC concentrations (right).

FIG. 13 is a graph of neurite length as a function of chondroitin sulfate to collagen dry weight ratio.

Typical human eyes have the lens and the iris. The posterior chamber is behind the iris and the anterior chamber is located before the iris. The eye has a cornea consisting of five layers as discussed herein. One of the layers, the corneal epithelium, covers the outer front of the cornea. Corneal epithelium is a stratified squamous epithelium that extends laterally to the periphery.

The five layers of the cornea include corneal epithelium, Bowman's membrane, parenchyma, Desme's membrane and endothelium. Corneal epithelium typically has a thickness of about 5 to 6 cell layers (about 50 micrometers thick) and is generally regenerated when the cornea is damaged. Corneal epithelium provides a relatively smooth refractive surface and helps prevent eye infections. The corneal parenchyma is a laminate structure of collagen in which cells such as fibroblasts and keratinocytes are dispersed. The parenchyma accounts for about 90% of the corneal thickness. The anterior part of the parenchyma, located under the epithelium, is acellular and known as the Bowman's membrane. The Bowman's membrane is located between the epithelium and the parenchyma and is thought to protect the cornea from damage. The corneal endothelium is typically a monolayer of low-cubic or squamous cells that dehydrate the cornea by removing water from the cornea. Human adult corneas are typically about 500 μm (0.5 mm) thick and typically have no blood vessels.

Ophthalmic devices have been invented that provide one or more benefits to an individual, such as a human, who desires to improve or improve vision or needs treatment of diseases, disorders or trauma to the eye. The device described herein can be configured as a corneal onlay, corneal inlay or full-layer corneal implant. The device of the present invention may improve vision of or provide vision to an individual who has lost vision. The device described herein does not specifically comprise an intraocular lens.

As used herein, "optically clear" refers to a white light transmittance of at least 85%. In certain embodiments, “optically clear” refers to the optical clarity of a healthy cornea, eg, a cornea having greater than 90% white light transmission and less than 3% scattering.

As used herein, “corneal onlay” is an ophthalmic implant or device, eg, having such size or shape, configured to be located between the Bowel's epithelial or epithelial cell layer of an individual, such as a human or animal. In comparison, the contact lens is configured to be located on the epithelium of the eyeball. Thus, the corneal onlay may comprise one or more portions located throughout or extending into the Bowman's membrane. This portion occupies a small part of the area or volume of the device, for example less than 50%.

As used herein, “corneal inlay” is a device or implant configured to be located within the parenchyma of the eye. Corneal inlays can be placed in the parenchyma by forming flaps or pockets in the parenchyma. The corneal inlay is located under the Bowman's membrane of the eye.

As used herein, a "periosteal corneal implant" is a device configured to replace all or part of an unhealthy cornea of an eye located in front of an aqueous humor of the eye.

Ophthalmic devices of the present invention have reduced cytotoxicity or are non-cytotoxic and provide one or more advantages to individuals equipped with the device. For example, the device may include (i) the desired refractive index, (ii) the desired optical transparency (in the case of visible light, the transmittance and light scattering degree equal to or better than that of healthy human corneal material having the same thickness), ( iii) desirable optical function, such as vision enhancement, (iv) improved comfort, (v) improved corneal and epithelial health, and (vi) therapeutic benefits, such as the treatment of diseases, disorders or trauma of the eye Provides more. The ophthalmic device of the present invention is made of a transparent or transparent material. Some examples of such devices include optically clear devices.

(i) moldable to form a matrix with acceptable optical function, for example moldable, (ii) optically clear or visually transparent, and (iii) facilitating nerve growth through and / or on the device. By manufacturing the device with a material which is effective to achieve the above advantages, as well as other advantages can be obtained. If the device is corneal onlay, the device is effective to facilitate re-epithelialization on the front of the device.

The device is made of a material that has sufficient mechanical or structural properties to withstand handling, implantation, which may include sutures, and wear and tear after mounting. The device provides or allows for sufficient nutrient and gas exchange to maintain a healthy eye. Devices manufactured in a mold, such as a corneal onlay, are made of a material that can be molded into a suitable size and shape, including edge gradients and vision correction curvature, as discussed herein.

In one embodiment of the present invention, a vision enhancing ophthalmic device includes a body comprising a material effective to facilitate nerve growth through the body when the device is located in an eye of an individual. By facilitating nerve growth through the body, the cornea of the individual receiving the device maintains its contact sensitivity. The body is formed to have an optical function. Thus, the body can be understood as a lens body. As discussed herein, the device is configured to be a corneal onlay, corneal inlay or penetrating corneal implant, eg, having such size and shape. In certain embodiments, the refractive error correction apparatus of the present invention may not have an optical function. For example, it can be understood that a refractive error correcting device according to the present disclosure is a blank that can be located between the corneal epithelium of the patient and the Bowman's membrane or within the corneal parenchyma of the patient.

In the case of corneal onlays, the substances that form the onlays provide or permit the exchange of gas and nutrients (eg, glucose) between the Bowman's membrane and the epithelium, thereby maintaining a regenerating and fully functional epithelium. Other nutrients include factors or substances that promote or enhance the survival, growth and differentiation of cells such as epithelial cells. The exchange should be the same or better than that of healthy human corneas. Permeability of the substance to nutrients and / or drugs can be monitored using conventional techniques. In addition, the transport of nutrients and / or drugs through the material should not alter the optical properties of the material. The onlays or lentices are fully biodegradable and allow for rapid attachment of the epithelium to the onlays and allow for recovery of nerve domination and sensitivity, eg contact sensitivity.

The ophthalmic device of the present invention may comprise an extracellular matrix (ECM) component. In certain devices, the body material comprises collagen, consists essentially of collagen, or consists of collagen. Collagen can be crosslinked using, for example, EDC / NHS during the manufacture of the device. The amount of collagen provided in the hydrogel device of the present invention is greater than the amount of collagen currently used in other ophthalmic devices. For example, the amount of collagen provided in the device of the present invention is typically greater than 1% (w / w) or (w / v), as discussed herein. In certain embodiments, the amount of collagen is greater than 2.5%. For example, the amount of collagen may be about 5.0% or more. In certain embodiments of the device of the invention, the amount of collagen is about 1 to about 50% (w / w), for example 2.5 to about 50%. For example, the amount of collagen is greater than about 6% (w / w). Alternatively, the substance may comprise about 10 to about 30% (w / w) collagen. As is known to those skilled in the art, about 15% by weight of the hydrated human cornea is collagen (Maurice DM: The Cornea and Sclera, pp 489-600. The Eye, Vol I, Second ed., Ed. H. Davson.Academic Press, New York, 1969). Thus, the device of the present invention contains more collagen than the amount of collagen present in existing ophthalmic devices and much more similar to the amount of collagen present in the human cornea. The amount and type of collagen provided in the device of the present invention also provides the desired refractive index, the desired optical clarity, the formability, and is effective in handling, implanting, sealing the device to the eye, and withstanding wear and tear after mounting.

The remaining portion of the ophthalmic device, such as a non-collagen-based portion, may be a liquid such as water or saline, or may include one or more additional polymers such as biopolymers and the like. For example, as disclosed herein, an ophthalmic device comprising about 24% (w / w) collagen may comprise about 76% (w / w) of a liquid, such as water or saline. In other words, the ophthalmic device may, in the hydrated state, comprise a collagen component that is 24% of the weight of the hydrated ophthalmic device. As another example, the ophthalmic device may comprise a collagen component that is 24% of the weight of the hydrated device, and a second polymeric component that is 6% of the weight of the hydrated device, with 70% of the weight being liquid.

As will be appreciated by those of ordinary skill in the art, in a state where the device is not hydrated, the amount of collagen in the device may be a percentage greater than the amount of collagen in the device in the hydrated state.

Collagen contains three polypeptide chains and is helical. As used herein, the term "collagen polymer" refers to triple helix collagen molecules. Collagen is a rod-like molecule that has a length of about 300 nm and a diameter of about 1.5 nm. Collagen molecules have an amino acid sequence called "telopeptide" on both the N-terminus and C-terminus, including most of the antigenicity of collagen. Atelocollagen is obtained by pepsin digestion (see DeLustro et al., J Biomed Mater Res. 1986 Jan; 20 (1): 109-20) and has no telopeptides, and thus has low immunogenicity. (Stenzel et al., Annu Rev Biophys Bioeng. 1974; 3 (0): 231-53).

Collagen for use in the devices defined above may be obtained or derived from any suitable collagen source, including animal, yeast and bacterial sources. For example, the collagen may in particular be human collagen, bovine collagen, porcine collagen, bird collagen, mouse collagen, horse collagen, or may be recombinant collagen. Recombinant collagen used in the device of the present invention may have one or more structural or physical properties that are not present in the collagen obtained from normal animal sources, since the recombinant collagen is obtained from bacteria, yeast, plants or transgenic animals. Because it becomes. For example, recombinant human collagen can include different glycosylation components that are derived from an animal and may not be present in processed collagen. In addition, recombinant collagen may have a different degree of crosslinking than animal-derived collagen, which may vary in composition. Variation in the degree of crosslinking in animal-derived collagen can lead to undesirable variations in collagen and chemical and physical properties. Recombinant human collagen not only has strictly controlled purity, but is also not associated with virus and / or prion contamination that may be associated with animal-derived collagen. Collagen useful in the device of the present invention can be obtained formally or synthesized using conventional techniques. For example, recombinant collagen can be obtained from Fibrogen (prepared from mutigene yeast bioreactor medium) or Pharming (Netherlands) (produced from transgenic cattle or rabbit milk) or Recombinant collagen can be prepared and obtained using the methods disclosed in PCT Publication WO 93/07889 or WO 94/16570. In certain devices, the collagen may be type I collagen. The device may also be prepared as atelocollagen (eg collagen without telopeptide). In certain embodiments, the collagen is a type of collagen that is not denatured. Atelocollagen can be obtained from a company such as Koken Japan (Supplier A as described herein), where bovine collagen is 3.5% (w / v) in the neutral composition and 3.0% (in acidic composition) w / v) and 10% (w / v) in the acidic composition, and pig collagen is available as 3.0% (w / v) or acidic lyophilized pork collagen powder in the acidic composition. Acidic lyophilized pork collagen powder may also be obtained from Nippon Ham (Japan) (Supplier B as described herein). Becton Dickinson (source C as described herein) provides 0.3% acidic and 10% acidic collagen compositions.

Among some collagen types, atelocollagen I provides ease of dissolution, handling and clarity of the final device. Such collagen (bovine, swine or recombinant collagen, in neutral or acidic solutions, or as acidic lyophilized powder) is available from several companies as described above. Lyophilized acidic porcine collagen is readily soluble, so that stirring at 4 ° C. in cold water yields a homogeneous (non-milky white) aqueous solution of concentration up to 33% (w / v). The clear collagen composition, such as solution, has a pH of about 3 (source B) or about 5 (source A). Commercial acid collagen compositions at concentrations as low as 0.3% (w / v) are concentrated by vacuum evaporation with stirring at 0-4 ° C. to obtain a clear solution having a final collagen concentration of about 10% (w / v) or less. Can be used in the manufacture of the device of the invention.

By using collagen type I that has not been denatured (i.e., becoming gelatinous by losing all or a substantial portion of the triple helix structure) during isolation and purification, a relatively robust or strong ophthalmic device can be obtained.

Differential scanning calorimetry (DSC) is a useful technique for determining the quality of a collagen solution at a supplier based on its triple helix content (Table 1). For a nearly perfect triple helix content, the denaturation DSC enthalpy (ΔH _denaturation ) is 65 to 70 J / g (based on dry collagen weight). From the DSC data, the ΔH _denaturation results indicate that the commercial acidic lyophilized porcine collagen solution and some commercial bovine collagen solutions are complete triple helix structures.

Collagen solutions with a low triple helix content (ΔH _denaturation <5 J / g, source C, Table 1) have a relatively low viscosity and a collagen composition or solution with a triple helix content close to 100% of the same concentration. Provides a weak gel as compared to. Collagen compositions (solutions) with ΔH _denaturation greater than about 60 J / g have been found to provide acceptable ophthalmic devices.

Denatured enthalpy of collagen solution Commercial collagen samples Furtherance ΔH _denaturation (dry collagen J / g) Koken (Japan), supplier A 10% bovine collagen solution 65.3 Koken (Japan), supplier A 10% bovine collagen solution, concentrated from 3% acidic solution 67.5 Koken (Japan), supplier A 5% bovine collagen solution, concentrated from 3.0% acidic solution 66.4 Koken (Japan), supplier A 3.5% Neutral Bovine Collagen Solution 68.1 Koken (Japan), supplier A Heat Denatured 3.5% Neutral Bovine Collagen Solution 24.4 Koken (Japan), supplier A 3.0% Porcine Collagen Solution 72.0 Koken (Japan), supplier A 5% solution from lyophilized pig collagen (acid) 68.1 Nippon Ham, Source B 10% solution from lyophilized pig collagen (strongly acidic) 63.4 Becton Dickinson, source C 5% bovine collagen solution concentrated from 0.3% solution 59.4 Becton Dickinson, source C "10%" bovine collagen solution 4.8 Fibrogen Recombinant Human Collagen 10% solution concentrated from 0.3 wt / wt% 67.7

In certain embodiments, including the embodiments described above, the body material can comprise a crosslinked collagen polymer. Alternatively, in other words, the body material may comprise two or more crosslinked collagen polymers. For example, the body material may comprise a first collagen polymer, a second collagen polymer and a third collagen polymer. Other materials may include more than three collagen polymers. Crosslinked polymers can be understood as collagen components of ophthalmic devices.

Accordingly, the vision enhancing ophthalmic device according to the present invention may have collagen components of about 1 to 50% (w / w) and comprise collagen components formed to have optical function. As discussed herein, in certain embodiments, the amount of collagen is greater than 2.5%, for example at least about 5.0%. For example, in certain embodiments, the collagen is greater than about 6% (w / w). For example, the amount of collagen is about 10 to about 30% (w / w). For example, the amount of collagen may be about 10 to about 24% (w / w). In certain devices, collagen is the only water-expandable (eg hydrogel) polymer of the device. In other devices, the collagen may be the only device or lens-forming polymer. For example, the device may contain 100% collagen in a dry state. As discussed earlier. In certain devices, for example, EDC / NHS can be used to crosslink or at least partially crosslink the collagen.

Collagen polymers used in the preparation of the compositions and devices of the present invention may be derived from the same collagen source or from different collagen sources. Or, in other words, a single type of collagen, such as type I atelocollagen (containing a large number of collagen polymer chains), is processed in an effective manner to allow the collagen polymers to crosslink with each other. In one embodiment, the collagen polymer is recombinant collagen. In another embodiment, the collagen polymers are both from the same animal source. Individual collagen polymers in a single composition may have different molecular weights.

It will be appreciated that the refractive error correction apparatus of the present invention includes cross-linked recombinant collagen. The amount of collagen present in such devices is greater than the amount of other collagen in the previously described refractive error correction device. Such a device may be formed to have an optical function.

The device disclosed herein is transparent. For example, the device should be optically clear. For example, the device should provide a minimum light scattering degree (equivalent to or better than the light scattering of healthy human corneal tissue) when placed in the eye of an individual. In addition, the device disclosed herein has a refractive index. In certain embodiments, the refractive index is about 1.34 to about 1.37. For example, the refractive index may be 1.341 to 1.349. When the device is configured as a corneal onlay or corneal inlay, the device is placed in a healthy eye of the individual as compared to the cornea being configured to be located in a damaged or diseased eye where a full corneal implant may be required. It is composed. In certain embodiments of the device of the invention, the device does not have a yellow group or yellow color. For example, the device may be designed to reduce or eliminate yellow or yellow that may be associated with some collagen-containing compositions.

The device of the present invention has a front side and a rear side. Thus, the body or collagen component of the device may have a front side and a back side. The front and back are generally opposite surfaces. The front side of the device refers to the surface that rests the retina when the device is located in the eyeball, and the back side faces the retina when the device is located in the eyeball. If the device is corneal onlay, the backside may be adjacent to or in contact with the Bowman's membrane and the front side may be adjacent to or in contact with the corneal epithelium. If the device is a corneal inlay, the front face will be in the parenchyma so that the front face is adjacent to or facing the Bowman's membrane and the back face toward the retina side of the eye. If the device is a penetrating corneal implant, the front side may face toward the corneal epithelium and the back side may be adjacent to or in contact with the corneal endothelium.

The device of the present invention may further be surface modified, or the device of the present invention may be surface modified to affect cell growth and / or differentiation on one or both of the front and rear surfaces. For example, corneal onlays may not be applied to surface modifications that affect cell growth on the front or back. As used herein, "cell growth" refers to the increase in cells or cell numbers. Thus, cell growth refers to the physical growth of individual cells, such as increases in surface area, volume, etc., proliferation of cells, for example cell division, and in some cases forming layered multilayers as found in healthy human corneas. Refers to the movement of cells. Cell growth refers to the growth of neurons, such as the extension of one or more neuronal processes on or under the device or through the device, and the growth or migration or proliferation of epithelial or endothelial cells on the surface of the device. As used herein, "cell differentiation" refers to the morphological, biochemical and physiological changes that single or multiple totipotent, multipotent or immature progenitor cells (including stem cells) undergo to achieve the final phenotype. Say. In certain embodiments of the device of the invention, epithelial cells grow on corneal onlays and are tightly coupled thereto, for example, directly on the front of the onlays, in particular onlays.

In certain corneal onlays, the body or collagen component is rearranged to be effective to reduce epithelial cell growth under onlays when the onlay is located within the eye of the individual. Additionally or alternatively, the corneal onlay is a body or collagen component that has been modified to be effective to promote epithelial cell growth (including migration) on the front of the onlay when the onlay is located within the eye of the individual. It may include. In this regard, the penetrating corneal implant may comprise a body or collagen component that is rear modified to be effective to reduce endothelial cell growth on the back of the penetrating corneal implant when the implant is located within the eye. The penetrating corneal implant may not be fully modified.

Examples of surface modifications that can reduce cell growth include providing a plasma-polymerized fluorinated monomer film, such as CF ₄ or C ₃ F ₈ , on one or both of the front and back sides, one or both of the surfaces Providing low free surface energy to the phase, and / or making one or both of the surfaces hydrophilic. By providing an alginate coating on one or both of the surfaces, the surface can be made hydrophilic.

The device may include one or more cell growth promoters that facilitate cell growth on or through the device. In certain embodiments, the cell growth promoter comprises a peptide. For example, the cell growth promoter may be a peptide having an amino acid sequence comprising RGD, YIGSR or IKVAV. Type I collagen itself is a rich source of RGD sequences. In certain embodiments, the cell growth promoter is the bioactive or neurotrophic portion of a neurotrophic factor or molecule. For example, the neurotrophic factor may be nerve growth factor (NGF), epidermal growth factor (EGF or HB-EGF) or basic fibroblast growth factor (bFGF or FGF-2). The cell growth promoter can be formed integrally with the collagen component or the body of the device, in other words, the cell growth promoter can be provided substantially throughout the device. In comparison, some ophthalmic devices include peptides provided only on one surface of the device.

In certain embodiments, the collagen-based ophthalmic device comprises a collagen component processed at an acidic pH during device fabrication. Acidic pH is particularly useful when the collagen component comprises a first collagen polymer crosslinked to a second collagen polymer. The acidic pH used in the manufacture of such devices is typically less than about 6.0, for example the pH may be from about 5.0 to about 5.5. Maintaining acidic pH and preventing or reducing pH surge during pH adjustment reduces fibrillogenesis of collagen. In addition, keeping the pH above about 5.0 does not degrade collagen as quickly as when the pH is below 5.0.

Any small or polymeric collagen-reactive substance or molecule can be used to crosslink the collagen polymer. In crosslinking chemistry, it is possible to use conventional methods or novel drugs commonly known to those skilled in the art. By crosslinking the collagen polymer, the device can maintain optical clarity and withstand biodegradation.

In certain embodiments, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC; CAS # 1892-57-5) and N-hydroxysuccinimide (NHS) are used to crosslink the collagen polymer Combine. In other words, the crosslinking agent used in the manufacture of the device is EDC / NHS. The collagen polymer and the EDC / NHS crosslinker are mixed together at acidic pH while preventing pH surge. After sufficient mixing, a portion of the mixed composition is placed in a mold and allowed to cure in the mold, forming an ophthalmic device. One of the advantages of using water soluble EDC / NHS to crosslink collagen and CSC is the formation of zero length (amide) bonds. This reduces the chance of grafted toxic substances into the tissue. In addition, since unreacted drugs and by-products of the EDC / NHS reaction are water soluble, they can be easily removed after gel formation.

In certain embodiments, the crosslinking agent or crosslinking agent with reduced cytotoxicity is used to crosslink the collagen polymer. Such crosslinkers preferably do not cause irritation or side effects when the ophthalmic device is placed in the eye of an individual. In some embodiments, the crosslinker is a crosslinker other than glutaraldehyde. Although glutaraldehyde may be a useful crosslinking agent in certain embodiments, glutaraldehyde may be undesirable because of handling and safety requirements.

In a further embodiment, the process further comprises poly (N-isopropylacrylamide-co-acrylic acid), chondroitin sulfate, keratan sulfate, dermatan sulfate, elastin, chitosan, which can be mixed with the collagen composition. And using one or more of N, O-carboxymethylchitosan, hyaluronic acid, hyaluronic acid aldehyde and alginate. Thus, the ophthalmic device may comprise one or more non-collagen polymers comprising a collagen component, for example a matrix of crosslinked collagen polymers, and a biopolymer. Non-collagen polymers may be crosslinked together and / or crosslinked to collagen polymers to form a network or matrix of crosslinked polymers.

In certain embodiments, the compositions are mixed together through relatively narrow channels or passageways to induce strong shear between different compositions. In one embodiment, the composition is mixed using a syringe system. Mixing relies on pumping syringes through narrow channels leading to strong shear between the viscous collagen solution and the drug. The channel diameter is chosen to suit the viscosity and burst strength of the syringe or other similar device. For high viscosities (for example 20-30% (w / v) collagen solution), low volume syringes with small diameter syringe plungers are used because higher pressures can be achieved using hands. Mixing is performed at acidic pH (eg about 5.0 to about 5.5) and low temperature (eg about 0 to about 5 ° C.).

Compared to other ophthalmic devices, the device of the present invention is made without using living cells. Accordingly, the inventors of the present invention have invented a novel method for preparing compositions and ophthalmic devices containing relatively high, near physiological concentrations of collagen without the use of live corneal cells. In addition, the device of the present invention contains substantially or no synthetic dendrimer component used to increase the cross-reactivity of collagen in other devices.

Additional neuro-friendly materials can be used in the manufacture of the device of the invention. Such materials are prepared by the methods disclosed herein and tested for neuro-affinity, such as nerve growth, using conventional methods commonly known to those of ordinary skill in the art, such as cell culture systems and the like. For example, materials can be tested and identified using the method disclosed in WO 2004/015090, filed August 11, 2003.

The device disclosed herein is configured to be positioned in the eye around the corneal region of the eye, for example, having such size and shape. If the device is a corneal onlay, the onlay may have a diameter of about 4 to about 12 mm, for example about 6 mm. The onlay may have an edge thickness of less than about 30 μm, for example between about 10 μm and about 30 μm. The onlay may have a center thickness of about 70 μm.

Onlay shaped molds can be made from polypropylene, which can have a diameter of 4 mm, 6 mm, 8 mm or 12 mm. The mold should be relatively stiff (for example, not bent while closed) and transparent to show the filling. The mold is configured to provide an onlay edge that is finely tapered (eg about 10 μm), or a slightly steep (eg about 30 μm) onlay edge.

The corneal implant template (front layer or partial layer) may have a diameter of about 12 mm. The implant mold is shaped to have the desired curvature and thickness of the cornea. If necessary, an ophthalmic device (eg hydrogel) may be trephined as needed during the implantation process.

One example of the refractive error correcting apparatus of the present invention is shown in Figs. 8 and 8A.

The corneal onlays disclosed herein may be configured to correct one or more wavefront aberrations of an individual's eye. Information on wavefront technology and measurement of wavefront aberrations is provided in US Pat. Nos. 6,086,204 to Magnate and WO 2004/028356 to Altmann. By shaping the mold into a shape necessary to cause the onlay to have a shape suitable for correction, the corneal onlay can be shaped to be suitable for correction of wavefront aberration. A method of using wavefront aberration measurements in corneal onlays is disclosed in US patent application Ser. No. 60 / 573,657, filed May 20, 2004. The onlay may be ablated to suit the correction of wavefront aberration. For example, laser or laser-like devices, lathes, and other suitable lens shaping devices may be used to ablate the onlays.

The corneal onlays disclosed herein may comprise a number of different bands. For example, the corneal onlay may include an optical band and a peripheral band. Typically, the optical band is surrounded by a peripheral band, that is to say, the optical band is generally located about the optical axis of the onlay (e.g., the central optical axis), the peripheral band being around the edge and periphery of the optical band of the corneal onlay. It is located between the edges. Depending on the particular deterioration of vision experienced by the patient, additional bands and onlay structures may be provided to the onlay.

In addition, the corneal onlays of the present invention may have non-boundary bands, for example two or more bands that do not have a visually or optically detectable boundary. The bands of the onlay may be smooth and continuous, and the onlay may be optically optimized to correct not only for refractive errors, but also for or apart from correction of refractive errors, as well as other optical aberrations of the eye and / or optical device. As will be appreciated by those skilled in the art, corneal onlays can be configured to correct macular degeneration, including but not limited to myopia, hyperopia, astigmatism and presbyopia. Onlays may use one or both of optical or physical means provided on the parenchyma of the eye to enhance or improve vision loss. Thus, the corneal onlay may be a monofocal lens or a multifocal lens including but not limited to bifocal lenses.

Additionally or alternatively, the corneal onlay may be a toric lens. For example, the onlay may include toric regions that may be effective to correct or reduce astigmatism when placed on an eye with astigmatism. The onlay may comprise a torus region located on the backside of the onlay or the onlay may comprise a torus region located on the frontside. Advantageously, toric onlays can be used without a ballast that keeps the onlays properly positioned on the eyeball because the onlays can be fixed in a relatively fixed position by the device epithelium. However, ballasts can be provided if desired. In certain embodiments, the onlay may include a ballast, such as a prism, or may include one or more thinned regions, such as one or more inferior and / or thin zones of a superior. In onlays configured to correct presbyopia, the onlays may employ one or more designs, such as concentric, aspherical (with positive and / or negative spherical aberration), diffractive and / or multi-zone refraction. It may include.

The invention also includes compositions such as synthetic or non-natural compositions. The composition can be fully or partially synthetic. For example, the present invention relates to an optically clear composition. Such compositions can be used in the manufacture of one or more ophthalmic devices disclosed herein. Alternatively, the composition may be used in a non-ophthalmic device as a non-ophthalmic composition or may be used in an ophthalmic device and may not provide a refractive error correction effect. In another embodiment, a composition according to the present disclosure comprises more than about 1% (w / w) collagen in a hydrated state and is optically clear. As discussed herein, the amount of collagen may be greater than 2.5%, for example greater than about 5.0%. For example, the composition may comprise about 1 or 2.5 or about 5.0 to about 30% (w / w) collagen in a hydrated state. In certain embodiments, the composition may comprise about 6% (w / w) collagen. In another embodiment, the composition may comprise about 10 to about 24% (w / w) collagen. The composition may comprise more than about 1% (w / w) crosslinked collagen in the hydrated state, crosslinked by EDC / NHS.

The composition of the present invention may comprise two or more collagen polymers. In certain embodiments, the composition comprises a first collagen polymer crosslinked to a second collagen polymer as described above. The composition may comprise substantially or no cytotoxic substances, such as glutaraldehyde.

The ophthalmic device disclosed herein can be placed in the eye using any suitable method or technique.

For example, by removing or separating a portion of the epithelium from the Bowman's membrane, corneal onlays can be placed on the Bowman's membrane of the eye. In certain situations, an alcohol, such as ethanol, may be applied to the corneal epithelium in a specific amount to remove the epithelium from the eye. The alcohol may be at a concentration of about 10 to about 60%, for example about 20 or about 50%. Warming ethanol to about 37 ° C. (eg body temperature) may be effective to promote epithelial removal. This epithelial removal technique is similar to the LASEK technique currently being implemented.

In another situation, the onlay may be placed on the Bowman's membrane by placing the corneal onlay under the epithelial flap or within the epithelial pocket. Such flaps and pockets can be made using cutting devices, blunt tools, and the like. Examples of methods for positioning corneal onlays in the eye are disclosed in US Application No. 10 / 661,400, filed September 12, 2003 and US Application No. 60 / 573,657, filed May 20, 2004.

Corneal inlays can be placed in the eye by forming a parenchymal pocket or corneal flap and placing the corneal inlay in or under the flap.

The penetrating corneal implant can be placed in the eye by removing the damaged or diseased portion of the cornea and placing the corneal implant in or near the area of the removed portion of the cornea.

Forceps or any other suitable inserter as described in US Patent Application No. 10 / 661,400, filed September 12, 2003 and US Patent Application No. 60 / 573,657, filed May 20, 2004. Thus, the ophthalmic device disclosed herein can be placed in the eye.

To facilitate positioning of the ophthalmic device within the eyeball, the device may include a visualization component. The visualization component can be any suitable element that makes the device easily visible while the device is inserted or positioned in the eye. For example, the visualization component may include one or more markings that may aid in rotational placement of the device, and the visualization component may include dyes or colorants, such as biodegradable or non-cytotoxic dyes.

Further details relating to the ophthalmic device of the present invention and methods of making and using such devices are provided in the following examples, which illustrate but do not limit the invention.

Example 1

Preparation of Collagen-Based Corneal Onlays

Typically, 0.5-2.0 ml of collagen solution in aqueous buffer was mixed with 0.01-0.50 ml of crosslinker in aqueous buffer at about 0 ° C. without air bubble entrapment. In some compositions, a second biopolymer other than collagen was added to the composition.

To mix the composition, a syringe containing the composition is connected to a Tefzel Tee-piece (Uptight Fittings) to control without thorough mixing and / or pH surge of viscous collagen solution It forms a micro-manifold that allows for neutralization. pH surges often result in irreversible fibroblast formation of collagen, providing an opaque matrix.

More specifically, a first luer adapter is used to hold the septum cut to size to fit the bottom of the threaded hole of the tee piece. The bulkhead was a cut of the "Ice Blue" 17 mm general purpose 22397 bulkhead from Restek Corporation. A first syringe containing a buffer such as MES (2- [N-morpholino] ethanesulfonic acid) buffer was placed in a second luer adapter and any air bubbles were pushed along with the buffer. The collagen solution was placed in a second syringe and connected to a third luer adapter of a Tefgel T-fragment (as shown in FIG. 1) equipped with three luer adapters (FIG. 2). The complete assembly is shown in FIG. 3.

Collagen solution by repeatedly pumping between the first and second syringes through the tee-fragments such that fluid flowing through the narrow hole channels (e.g. about 0.5 to about 0.25 mm) in the tee-fragments strongly shears the liquid. Was mixed thoroughly with MES buffer. The pH was adjusted to 5.0 to 5.5. These were then mixed by passing the collagen / buffer mixture and EDC and NHS solutions (molar equivalent ratio of EDC: NHS 1: 1) at 0-4 ° C. through another manifold using another syringe.

Droplets of each substantially homogeneous solution are immediately dispensed into an onlay mold and cured for 5 to 24 hours (eg 15 hours) at room temperature first in a 100% humidity environment and then 15 to 24 hours at 37 ° C. Curing.

Each final onlay sample was immersed in phosphate buffered saline (PBS) for 2 hours and then carefully separated from its template.

In some cases, such gels can be impregnated with an aqueous solution of a second reactive biopolymer to further crosslink and add new biological factors.

Finally, the crosslinked onlay hydrogel was impregnated in PBS solution (containing 0.5% in PBS, 1% chloroform) at 20 ° C. to terminate any reactive residue and extract the reaction byproducts. This sterile equilibrium hydration onlay was thoroughly rinsed with PBS before all tests.

For gels prepared from several collagen / EDC-NHS solutions at higher collagen concentrations (10% or higher), the gel is first impregnated in pH 9.1 buffer to terminate any residual reaction and before storing in chloroform-saturated PBS The product was extracted properly. This basic extraction eliminated epithelial cytotoxicity problems with these samples. For many stoichiometry, sterile, cytotoxic gels were obtained by impregnating chloroform-saturated PBS and then removing the chloroform residue.

Example 2

Ophthalmic device containing cell growth promoter

Cell growth promoters, such as pentapeptides (YIGSR, active units in laminin macromolecules) alone or in synergistic peptides, such as those containing IKVAV, synergistic IGF, and substances that promote epithelial health The peptide, EGF, NGF, FGF, or some of these molecules, can be introduced into any collagen / EDC-NHS crosslinked device, including those having a second EDC-NHS reactive biopolymer. In the case of YIGSR, this cell growth promoter coupling can be achieved through the reactivity of the free amine end groups of the tyrosine residues on the promoter. Thorough extraction can be performed after gelation to remove any unbound cell growth promoter.

Details regarding specific combinations of ophthalmic devices are provided in Examples 3 to 13 and Table 2 below.

Example 3

1-ethyl- in MES buffer at pH 5.5, temperature maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and at an EDC: NHS molar equivalent ratio of 1: 1. An ophthalmic device was prepared as described in Example 1 using 3- (3-dimethylaminopropyl) carbodiimide (EDC) / N-hydroxysuccinimide (NHS) + collagen.

Example 4

COP + EDC- under conditions of pH 5.5, temperature at 4.8 buffer, starting at 0-4 ° C., maintained at 21 ° C. for 15 hours and then maintained at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. Using NHS + collagen, an ophthalmic device was prepared as described in Example 1. [Poly (N-isopropylacrylamide-co, a copolymer of COP) by free radical polymerization of NiPAAm and AAc in 1,4-dioxane with a 2,2'-azobis-isobutyronitrile initiator under nitrogen at 70 ° C Acrylic acid).

Example 5

EDC-NHS + at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. An ophthalmic device was prepared as described in Example 1 using chondroitin sulfate C (ChS) + collagen.

Example 6

Collagen + EDC-, at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. An ophthalmic device was prepared as described in Example 1 using NHS + N, O-carboxymethylchitosan (CMC).

Example 7

In MES buffer, pH 5.5, starting at 0-4 ° C. and maintained at 21 ° C. for 2 hours, the gel was secondly crosslinked by impregnation of chitosan (1% aqueous solution, 5000 Da) in PBS for 4 hours, and finally Collagen + EDC-NHS + N, O-carboxymethylchitosan (CMC), described at Example 1, at a temperature maintained at 37 ° C. for 15 hours and at an EDC: NHS molar equivalent ratio of 1: 1. An ophthalmic device was prepared as described.

Example 8

Collagen + EDC-, at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. Using NHS + hyaluronic acid (HA), an ophthalmic device was prepared as described in Example 1.

Example 9

Collagen + EDC-, at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and at an EDC: NHS molar equivalent ratio of 1: 1. An ophthalmic device was prepared as described in Example 1 using NHS + Chondroitin Sulfate (ChS) + Hyaluronic Acid (HA).

Example 10

Collagen + Hyaluronic Acid Aldehyde (HA-CHO) + Sodium Cyanoboro, at a temperature of pH 7-8, 0-4 ° C., 15 hours at 21 ° C. and 15 hours at 37 ° C. in PBS Using hydride, an ophthalmic device was prepared as described in Example 1. HA-CHO was prepared by oxidatively digesting HA (0.1 g) with sodium periodate (0.05 g) at 21 ° C. for 2 hours. The aqueous solution was dialyzed against water for 2 days.

Example 11

Collagen + EDC-, at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. Using NHS + alginate, an ophthalmic device was prepared as described in Example 1.

Example 12

In MES buffer, pH 5.5, starting at 0-4 ° C. and maintained at 21 ° C. for 2 hours, under conditions of secondary crosslinking by impregnating the gel with chitosan (1% aqueous solution, 5000 Da) in PBS for 4 hours, An ophthalmic device was prepared as described in Example 1 using glutaraldehyde (“Glut”, diluted 1% in water) + collagen. The gel in the template was warmed to 37 ° C. for 15 hours before being removed from PBS.

Example 13

Collagen + EDC-, at a temperature of pH 5.5, in a MES buffer starting at 0-4 ° C. and maintained at 21 ° C. for 15 hours and then at 37 ° C. for 15 hours, and an EDC: NHS molar equivalent ratio of 1: 1. Using NHS + chitosan, an ophthalmic device was prepared as described in Example 1.

Example 14

EDC- at conditions of pH 7-8, 0-4 ° C. and 15 h at 21 ° C. followed by 15 h at 37 ° C., and an EDC: NHS molar equivalent ratio of 1: 1 in PBS buffer. An ophthalmic device was prepared as described in Example 1 using NHS + Chondroitin Sulfate C (ChS) + Collagen.

All of the devices of Examples 3 to 14 provide a robust, clean, flexible gel having both the commercial collagen and reactant ratios specified in Table 2 below.

Some hydrogels for onlay applications have been characterized by DSC, optical isotropy and refractive index, measurements, tensile properties (stiffness, maximum tensile strength, elongation at break, Table 2) and in vivo performance. DSC measurements on the gel after the reaction for all examples showed an increase in denaturation temperature and a decrease in ΔH _denaturation , consistent with the crosslinking of collagen. The refractive indices of all formulations in Table 2 were 1.341 to 1.349.

Example 15

In vitro onlay performance (Table 2)

Using the method disclosed in Li et al., PNAS 100: 15346-15351 (2003), how epithelial cells (human immortalized corneal epithelial cells, HCEC) grow and reach confluence on hydrogels Were evaluated (how to reach confluence), how HCEC cells stratified on the hydrogel, and chick dorsal ganglion nerve growth on the hydrogel and into the hydrogel (if the data is valid, the latter is micron / day growth). Recorded as).

The human cornea restores the epithelium three to five days after complete removal.

In vitro test periods were typically about 6 to 8 days, but better formulations allowed re-epithelialization to reach confluence within 3 to 5 days or less. For denser gels (> 5% collagen), excellent over nerve growth (300 microns) was observed in in vitro tests for many formulations. In-gel nerve growth slowed rapidly with increasing gel stiffness but was observed by in-depth microscopy.

Hydrogel Composition and Performance Example No. Collagen Sources (Table 1)
(Initial concentration, wt / vol%) Collagen / XL Equivalence Ratio or (wt / wt) Final collagen concentration in the gel
(w / v%) M stress,
g power ^* Elongation at break
(Mm) ^* Stiffness s,
g / mm ^* In vitro epithelial cells, time to reach confluence In vitro nerve growth within 6 days ^** 2 B, AFDP:
(Soluble at 10%) Col-NH ₂ : EDC = 5: 1
Col: YIGSR
= 5: 0.0001 7.2 3-5 Gel: Fast
In gel: 27 μm / d 3 A, (10% small) Col-NH ₂ : EDC = 5: 1 7.3 8.0 2.6 4.0 3 B, AFDP:
(Soluble at 10%) Col-NH ₂ : EDC = 5: 1 7.3 9.7 4.0 4.4 2-3 Gel: Fast
In gel: 40 μm / d 3 B, AFDP:
(Soluble at 15%) Col-NH ₂ : EDC = 5: 1 10.8 13.08 4.6 3.0 2-3 3 B, AFDP:
(Soluble at 20%)
350㎛
Thickness of gel Col-NH ₂ : EDC = 10: 1 14.3 11.95 4.3 3.0 2-3 3 B, AFDP:
(Soluble at 32%) Col-NH ₂ : EDC = 1: 1 18.0 14 2-3 Gel: Fast
In gel: 30 μm / d 3 A, (3.5% neutral cow) Col-NH ₂ : EDC = 1: 1 2.7 3.1 2.0 1.7 3-5 Gel: Fast
5 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: ChS = (9: 1) 2.7 2.5 1.8 1.4 3 Gel: Fast
In gel: 41 μm / d 5 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: ChS = (4: 1) 2.7 2.7 1.8 1.5 3 Gel: Fast
In gel: 73 μm / d 5 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: ChS = (3: 1) 2.7 3.1 1.5 1.5 3 Gel: Fast
In gel: 70 μm / d 6 A, (3.5% neutral cow) Col-NH ₂ : EDC = 1: 1
Col: CMC = (1: 0.5) 3.6 1.6 2.0 6 B, AFDP:
(Soluble at 32%) Col-NH ₂ : EDC =
1: 1.3
Col: CMC = (15: 1) 1.45 6.0 3 7 A, (3.5% neutral cow) Col-NH ₂ : EDC = 1: 1
Col: CMC = (2: 1) + Soluble Chitosan 2.9 1.6 1.9 8 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: HA = (9: 1) 2.2 2.5 2.2 1.08 3-5 8 A, (5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: HA = (4: 1) 2.2 2.4 2.2 2.07 3-5 8 A, (10% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: HA = (3: 1) 2.2 2.0 1.8 1.13 3-5 9 A, (3.5% neutral cow) Col-NH ₂ : EDC =
0.5: 1.0
Col: HA: Chs =
9: 1: 1 2.3 3.0 1.7 1.7 3-5 Gel phase and intragel growth 10 A, (3.5% neutral cow)
350㎛
Thickness of gel Col-NH ₂ : HA-CHO =
1: 1 3.2 1.0 0.7 11 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: Alg = 4: 1 2.7 3.0 2.0 1.6 3-5 Gel: Fast
In gel: 41 μm / d 11 A, (3.5% neutral cow) Col-NH ₂ : EDC = 2: 1
Col: Alg = 2: 1 2.7 3.4 2.5 1.5 3-5 Gel: Fast
In gel: 13 μm / d 12 A, (3.5% neutral cow) Col: Glut =
(130: 1) + Soluble Chitosan 3.1 2.1 1.5 13 B, (11% AFDP), 900 μm thick gel Col-NH ₂ : EDC =
0.33: 1.0
Col: Chitosan =
(15: 1) 5.8 8.32 3.29 2.57 4 13 B, (11% AFDP), 500 μm thick gel Col-NH ₂ : EDC =
0.66: 1.0
Col: Chitosan =
(15: 1) 5.8 4.25 4.02 1.32 13 B, (11% AFDP), 900 μm thick gel Col-NH ₂ : EDC =
0.66: 1.0
Col: Chitosan =
(15: 1) 5.8 8.46 5.23 2.17

^† Abbreviation: Col collagen; Glut glutaraldehyde; HA hyaluronic acid; Chs chondroitin sulfate C; Free amine portion of Col-NH ₂ collagen; AFDP acid lyophilized pigs; epi. epithelium; ND Not determined.

^* 500 μm thick, 12 mm diameter implants unless otherwise noted. Stress, elongation and stiffness data were obtained from a suture pull out method as disclosed in Lee et al. (PNAS 100: 15346-15351 (2003)).

^** gel phase: neurites derived from DRG overgrowth hydrogels; In gel: neurites growing into hydrogel by the indicated length within 6 days

Example 16

In vivo onlay performance

Onlays were prepared as described in Example 1. A first set of onlays were prepared from 10% (w / v) porcine collagen and EDC / NHS. A second set of onlays was prepared from 3.5% (w / v) bovine collagen and chondroitin sulfate (CSC) and EDC / NHS. The onlay had a diameter of about 6 mm, a central thickness of about 70 μm, and an inclined edge of 30 μm.

To implant the onlays, the pig epithelium was treated with 45% ethanol for 30-45 seconds. An incision was made in the form of a butterfly and a pocket formed in the epithelium. For visualization, the onlays were stained with a blue cell-free dye (Gel-Code®). The pre-stained onlay was inserted into the pocket. Protective contact lenses were closed on the eye.

Visual tests were performed to assess inflammation, hyperemia and / or vascular infiltration of the cornea. Slit lamp microscopy was used to evaluate corneal clarity. A tonometer was used to measure intraocular pressure. Cochet-Bonnet aesthesiometer was used to determine corneal contact sensitivity. Contact sensitivity can be useful for assessing the presence of functional neurons, which has been demonstrated by in vivo confocal imaging and immunohistochemistry of harvested implant-containing corneas. Corneal topography was examined using the PAR Corneal Topographic System (CTS) immediately before implantation and three weeks after surgery.

Corneal topography was examined by aligning the eyes of anesthetized pigs to CTS. Dilute solutions of fluorophores and artificial tears were applied to the eye to coat the corneal surface and visualize the target grid. The device focal plane was adjusted so that the target grating was in focus with the focus on the cornea front. A digital image of the grid was captured. Digital images were analyzed to obtain measurements of the shape of the cornea front. By comparing the digital images before and after implanting the onlay, it is possible to evaluate the changes in the corneal shape due to the mounting of the onlay.

In vivo confocal microscopy can capture images of different depths in the cornea of a living pig, allowing monitoring of the eye's response to the ophthalmic device. For example, confocal microscopy can be used to monitor the presence of nerves in the device. In vivo confocal microscopy was performed by examining anesthetized pigs with a Nidek Confoscan 3 in vivo confocal microscope before implantation of the ophthalmic device and three weeks after surgery. Artificial tears were deposited on the eye to be examined. To reduce eye movement, two drops of topical anesthetic were applied to the eye. A confocal lens (gel impregnation) was contacted with the cornea along with a one layer gel on the front of the lens for refractive index matching. The device focal plane was adjusted to focus the corneal endothelium, and then an image of the cornea was acquired while the focal plane of the lens was scanned to the same depth as the corneal thickness.

In addition to histopathological examination of tissue sections stained with hematoxylin and eosin (H & E), immunohistochemistry was used to determine early signs of corneal epithelial recovery on onlays and attachment and interaction with underlying onlays. . Immunohistochemistry could also be used to demonstrate the presence of nerves and any infiltration of immune and inflammatory cells. Using conventional techniques, anti-neurofilament staining was performed on half the cornea, with or without permeabilization with a detergent, followed by the presence or absence of the implanted onlay. Immunofluorescence was used to visualize bound antibodies.

As discussed earlier, the cornea with corneal onlay was well cured and remained optically clear with minimal or no redness or inflammation. There was no sign of vascular invasion. Normal intraocular pressure was observed. Postoperative cornea showed contact sensitivity. Topographic examination showed that implanted onlays could affect the change of corneal topography. Onlay resulted in a thickness change of about 50 μm at the central corneal ridge. Epithelium adhered well to onlay. In vivo corneal microscopy revealed excellent general corneal structures with subepithelial and parenchymal nerves and cells distributed from the epithelium to the endothelium. H & E stained frozen sections showed that the onlay was integrated with the host cornea. Immunohistochemistry demonstrated that the adhesion of cells in the onlay-transplanted cornea was little changed when compared to untreated cornea using staining with E-cadherin. Keratin 3 and E-cadherin staining were comparable to the control. Type VII collagen staining to fix the fiber and basement membrane complexes was less pronounced than staining of the control. Staining for α6 integrins showed localization of basal epithelial cells in both engineered and untreated controls. Anti-nerve microfiber 200 antibody staining showed the presence of nerves in the implantation site in the cornea equipped with onlay. Anti-CD 45 antibody staining showed no inflammatory or immune response.

Example 17

Ablation of the corneal onlay

Collagen / EDC and collagen / chitosan onlays were ablated using a VISX Star S4 excimer laser (Table 3). Surface topographical measurements of onlays before and after treatment were obtained using the PAR Corneal Topographic System (CTS). For treatment, the onlay was removed from the storage solution and placed on a spherical surface made of PMMA.

Phototherapeutic keratectomy (PTK) provides a uniform number of laser pulses (or energy) over the entire ablation zone. Photorefractive keratectomy (PRK) changes the curvature as desired by changing the pulse density on the ablation zone. AZD is the ablation band diameter. Depth is the expected treatment depth on the human cornea as reported by the laser manufacturer.

Difference maps were generated from the preoperative and postoperative topographic maps of collagen / EDC onlay to demonstrate the effect of ablation.

PTK ablation was expected to provide a completely constant central blue area of about 5 mm in diameter (for tea maps). A small gradient of the amount of tissue removed was predicted because the onlay is a curved surface. Myopic PRK ablation was expected to eliminate the maximum tissue depth at the center. The depth of tissue removed was expected to gradually decrease to zero at the edge of the ablation. Primitive sphere correction was expected to remove tissue to the maximum at the edge of the treatment zone, leaving the center 1 mm diameter untouched, and expected to form outwards 9 mm from the center. After primitive correction, the secondary map was expected to show a blue ring around the central green band. The difference map obtained from myopic astigmatism correction was expected to look similar to the map obtained from myopic sphere correction, except that its blue pattern was elliptical.

The car maps obtained from ablation onlay showed the expected tissue removal pattern described above in all car maps. The maximum depth of tissue removed was expected to be larger than expected for the human cornea. For example, the rate at which corneal onlay material was removed was about 1.7 to about 2 times the rate of corneal removal. The difference between the rate of ablation and corneal removal of the onlay material was not uniform throughout the sample. The rate difference may be due to, among other factors, the depth of the process measurement, the material density and surface roughness, and the moisture content of the material.

Collagen / chitosan onlays were observed to ablate at a faster rate than collagen / EDC onlays. This difference may be due to hydration. For example, post-operative collagen / chitosan onlays may have a much lower moisture content than collagen / EDC onlays.

The ophthalmic device may comprise a strength enhancing component such as urethane.

Example 18

Human Recombinant Collagen Ophthalmic Device

Using a syringe system as described herein, 0.3 ml of 13.7 wt.% Human recombinant type I collagen and 0.3 ml of 0.625 M morpholinoethanesulfonic acid (MES) obtained from fibrogen, San Francisco, CA, USA, were mixed. Thereby, crosslinked collagen hydrogels were prepared. Mixing was carried out at low temperature by performing mixing in an ice water bath.

After obtaining a homogeneous solution, 57 μl of EDC / NHS were injected into the mixture such that the molar equivalent ratio to collagen free amine (coll-NH ₂ ) group was 3: 3: 1. To adjust the pH of the solution to about 5, NaOH (2N) was added to the mixture.

The mixture was poured into a glass or plastic mold and left for 16 hours at 100% humidity and room temperature. The template was then transferred to the incubator and post-cured at 37 ° C. for 5 hours.

This method was used to prepare other hydrogels containing human recombinant type I collagen in which the ratio of EDC / NHS to collagen coll-NH ₂ groups is 1: 1: 1 and 6: 6: 1.

Refractive index (RI) was determined on a VEE GEE refractometer. Transmittance was measured at wavelengths of white light, 450 nm, 500 nm, 550 nm, 600 nm and 650 nm. Direct tensile measurements such as stress, elongation at break and modulus were determined on an Instron electromagnetic tester (Model 3340). The size of the sample was 5 mm x 5 mm x 0.5 mm. The water content of the hydrogel was calculated according to the following formula:

(W-W ₀ ) / W%

Wherein W ₀ and W represent the weight of the dry sample and the expanded sample, respectively.

Human recombinant collagen hydrogel (denoted as F1) with a ratio (molar equivalent) of EDC / NHS / coll-NH ₂ having 1/1/1 had a refractive index of 1.3457 ± 0.0013. Human recombinant collagen hydrogel (denoted F3) having a ratio (molar equivalent) of EDC / NHS / coll-NH ₂ of 3/3/1 had a refractive index of 1.3451 ± 0.0002. Human recombinant collagen hydrogel (denoted F6) having a ratio (molar equivalent) of EDC / NHS / coll-NH ₂ of 6/6/1 had a refractive index of 1.3465 ± 0.0001.

Table 4 summarizes the light transmittances of the different hydrogels.

Light transmittance wavelength
(Nm) white light 450 500 550 600 650 Average light transmittance (%) F1 86.7 ± 0.9 69.7 ± 1.2 76.0 ± 1.3 79.2 ± 1.4 82.4 ± 1.3 84.9 ± 1.4 F3 90.7 ± 2.5 85.8 ± 3.5 86.4 ± 2.9 86.7 ± 2.6 88.0 ± 2.4 89.6 ± 2.6 F6 75.5 ± 1.5 48.7 ± 0.4 57.7 ± 1.1 62.7 ± 1.1 67.6 ± 1.4 71.6 ± 1.7

Hydrogel materials labeled F3 have been shown to exhibit the most acceptable optical properties. Visually or macroscopically, F3 has been shown to have the best transparency compared to other human recombinant hydrogels.

Table 5 provides the mechanical properties of the human recombinant hydrogels of the present invention.

Mechanical property Sample F1 F3 F6 Average maximum stress (KPa) 62.6 ± 9.9 117.2 ± 36.9 149.9 ± 57.7 Average Break Stress (KPa) 67.1 ± 21.0 110.5 ± 49.7 99.5 ± 60.8 Average Elongation at Break (%) 62.60 ± 6.82 50.20 ± 7.55 23.51 ± 9.03 Average Modulus (MPa) 0.281 ± 0.032 0.525 ± 0.124 1.949 ± 0.939

Hydrogel F3 has been shown to have a relatively low modulus but also other acceptable mechanical properties.

Table 6 provides the moisture content values suitable for hydrogel materials.

Equilibrium moisture content Sample F1 F3 F6 Water content (%) 92.82 ± 0.68 92.63 ± 0.61 91.40 ± 0.38

It is clear that the hydrogels are highly hydrated.

In this example, a pH indicator was added to the MES buffer to help monitor the pH change. The specific indicator used in this example is Alizarin Red S (Sigma Aldrich).

4 is a graph of human corneal epithelial cell growth on human recombinant collagen sample F1 over 7 days. 5 is a graph of human corneal epithelial cell growth on human recombinant collagen sample F3 over 7 days. 6 is a graph of human corneal epithelial cell growth on human recombinant collagen sample F6 over 7 days.

Cell growth observed on the recombinant hydrogel material was better than that observed in the control experiment.

7 is a photograph showing that hydrogel material F3 is maintained in vivo for at least 30 days.

Example 19

Collagen-Poly (NIPAAm-co-AAC) Composition

The composition was prepared using the EDC / NHS crosslinking method described herein (Example 4). Starting collagen concentration was 15%. Final collagen concentration was 11%. The final poly (NIPAAm-co-Aac) concentration was 3%. The total solid concentration in the gel was 14%.

Such materials, using the suture pull out method as disclosed in Lee et al., PNAS 100: 15346-15351 (2003), have a refractive index of 1.3542, a tensile strength of 11 g-force, an elongation of 3.3 mm and 3.8 g. Had a modulus of force / mm.

The denaturation temperature increased from 40 ° C. before crosslinking to 50 ° C. after crosslinking. The material had higher light transmittance and lower backscatter than the human or rabbit cornea. For example, when white light was used, the% transmittance was about 102% for hydrogel material, about 93% for human cornea, and 78% for rabbit cornea. The hydrogel had a% light transmittance of about 90%, 96%, 100%, 101% and 103% for wavelengths of light of 450 nm, 500 nm, 550 nm, 600 nm and 650 nm, respectively. Human and rabbit corneas showed less than 100%% light transmittance at all test wavelengths and consistently lower% light transmittance than hydrogel material at each wavelength.

The hydrogel material appeared to reach corneal epithelial cell confluence after 7 days of sowing.

Example 20

Collagen / Chondroitin Sulfate Composition

Chondroitin sulfate (CSC), and type I collagen, were used as proteoglycan equivalents to develop biosynthetic matrices with high optical clarity and tensile strength. Hydrogels containing up to 30% (w / w) CSC, based on collagen dry weight, were prepared under controlled conditions, without aggregation or collagen fibrosis, which could lead to loss of optical clarity. Hydrogels have been characterized physically and biochemically. In vitro tests showed that human corneal epithelial cells (HCES) grew well on the surface of the gel and stratified with success. The matrix helped good intracellular gel growth. Similar results were obtained in vivo.

The composition was prepared similar to Example 14 described herein. Using EDC and NHS, CSCs were covalently bound to collagen.

Collagen (3.5 w / v%) and CSC gels having different CSC to collagen dry weight ratios and different EDC to collagen-NH ₂ molar equivalent ratios were subjected to the EDC / NHS (1: 1 molar equivalent) crosslinking technique as discussed herein. Was prepared. All gels were visually clear. The composition had higher light transmittance and lower light scattering (about 87% light transmission and 3% backscattering) as compared to the human cornea, as indicated in Table 7.

Transmittance and Backscatter CSC to collagen weight ratio (%) 0 5 10 20 30 Light transmittance (%) 89.9 95.5 93.0 90.5 97.3 Backscattering degree (%) 0.30 0.19 0.19 0.17 0.19 EDC to NH ₂ ratio 0.25 0.5 1.0 2.0 Light transmittance (%) 96.7 100 99.9 88.2 Backscattering degree (%)  0.24 0.28 0.16 0.20

The refractive index was 1.34 to 1.35, which is similar to the refractive index of the human cornea (1.376).

The expansion ratio of gels prepared using different EDC to collagen-NH ₂ molar ratios was measured and this was calculated using the following formula:

Expansion ratio = (W _w -W _d ) / W _d

Wherein W _w is the weight of the hydrated gel and W _d is the weight of the dry gel.

Collagen-CSC crosslinking using EDC / NHS causes crosslinking between carboxylic acid and amine groups. This result (FIG. 9) suggests that as the EDC to collagen-NH ₂ ratio increases, the expansion ratio of the collagen-CSC gel decreases due to the introduction of a more compact network.

Measurement of the mechanical properties of the implant was performed using a suture pull out method as disclosed in Lee et al., PNAS 100: 15346-15351 (2003). The implant was fully hydrated in PBS and drawn at a rate of 10 mm / min. Tensile strength at rupture of the implant (500 μm thick and 12 mm in diameter) was monitored. The tensile strength of the gel was improved by increasing the EDC to NH ₂ molar ratio (FIG. 10). However, as the amount of EDC increased substantially, the material became vulnerable, so the tensile strength decreased slightly when the EDC to collagen-NH ₂ molar ratio was 2 or more.

By increasing the concentration of collagen as shown in Figure 11, it is possible to improve the tensile strength of the gel (using 10% collagen instead of 3.5% collagen). Tensile strength increased from 2.65 g-force to 10.02 g-force and the expansion ratio decreased from 21.5 to 12.1.

Crosslinking efficiency was evaluated by differential scanning calorimetry (DSC). Heating the collagen or crosslinked collagen hydrogel will induce structural variation of the original triple helix structure at a certain temperature depending on the nature and extent of the crosslinking. Collagen solution and crosslinked fully hydrated collagen hydrogels were characterized in a sealed pan, with the temperature of the sample rising at a constant rate of 2 ° C./min. The temperature at the maximum peak was recorded as the denaturation temperature. As the EDC to NH ₂ ratio increased, the denaturation temperature increased from 42.4 ° C. to 56.6 ° C. (FIG. 12A), suggesting that covalent bonds increased the stability temperature by increasing the stability of the triple helix. The denaturation temperature of the collagen-CSC gel was higher than the denaturation temperature of the gel composed only of collagen (FIG. 12B). However, changing the CSC to collagen molar ratio in the collagen-CSC gel did not change the denaturation temperature.

In vitro growth of human corneal epithelial cells from established cell lines was observed. Nerve growth was performed in vitro using the dorsal root ganglion implanted into the gel. Neurites were grown for 7 days, gels were stained for neurofibrils and neurite extension lengths were measured. Neurites grew well in all collagen-CSC gels (FIG. 13).

Increasing the concentration of CSC from 5% to 20% resulted in a significant improvement in the length of neurites extension in the gel. Additional benefits were not evident in gels containing 30% CSC (FIG. 13). Excellent epithelial coating and implant integration were observed.

Example 21

Type III collagen composition

Substances: Human recombinant type III collagen (5.1% w / w fibrogen incorporated), 0.625 M morpholinoethanesulfonic acid [MES, Alizarin Red S pH indicator (6.5 mg / 100 mL water)], 1-ethyl 3- (3-dimethyl aminopropyl) carbodiimide HCl (EDC), N-hydroxy-succinimide (NHS).

Hydrogels were prepared from 18.3% (w / w) type III collagen solution. 0.3 ml of 18.2% by weight human recombinant type III collagen (concentrated from 5.1% w / w human recombinant type III collagen fibrogen incorporated) and 0.3 ml of MES (0.625 M) were added to the plastic tea in the ice bath. Mix in two bubble free syringes. After the homogeneous solution was formed, 33.5 mg of EDC and 20.1 mg of NHS were dissolved in 0.125 ml of MES, 57 μl of which was taken and injected into the syringe at a molar ratio of 3: 3: 1 of EDC: NHS: collagen-NH ₂ . If the mixture appeared pink, the pH was about 5 and no NaOH solution was added. The mixture was mixed well and placed in a glass mold (thickness 434 μm) and left for 16 hours at 100% humidity and room temperature. The template was then transferred to the incubator and post-cured at 37 ° C. for 5 hours. The resulting flat hydrogel was taken out, impregnated in 10 mM PBS and replaced with fresh buffer every 8 hours. The obtained hydrogel was impregnated in 10 mM PBS containing 1% chloroform and stored in a 4 ° C. refrigerator.

Using the method described above, additional type III collagen hydrogels were also prepared having an EDC: NHS: collagen-NH ₂ ratio of 2: 2: 1 and 1: 1: 1. All gels obtained were clear.

Hydrogels were prepared from 5.1% (w / w) type III collagen solution. 0.3 ml of 5.1 wt% human recombinant Type III collagen and 50 μl of MES (0.625 M) were mixed in two bubble-free syringes connected to plastic tees in an ice bath. After a homogeneous solution was formed, 9.3 mg of EDC and 5.6 mg of NHS were dissolved in 0.125 ml of MES, 57 μl of which was taken and injected into the syringe in a molar ratio of 3: 3: 1 of EDC: NHS: collagen-NH ₂ . If the mixture appeared pink, the pH was about 5 and no NaOH solution was added. The mixture was mixed well and placed in a glass mold (thickness 434 μm) and left for 16 hours at 100% humidity and room temperature. The template was then transferred to the incubator and post-cured at 37 ° C. for 5 hours. The resulting flat hydrogel was taken out, impregnated in 10 mM PBS and replaced with fresh buffer every 8 hours. Finally, the obtained hydrogel was impregnated in 10 mM PBS containing 1% chloroform and stored in a 4 ° C. refrigerator. The resulting gel was optically clear.

The final collagen content for the 18.3% w / w collagen starting concentration is 8.36% (w / v) (calculated based on the dilution ratio after each component addition) or about 10% (w / v) (measured value) It was.

The final collagen content for the 5.1% w / w collagen starting concentration is 3.76% (w / v) (calculated based on the dilution ratio after addition of each component) or about 4% (w / v) (measured value) It was.

While the invention has been described in terms of various specific examples and embodiments, it will be appreciated that the invention is not limited thereto but other embodiments are within the scope of the invention.

Numerous publications, patents and patent applications have been mentioned above. Each publication, patent, and patent application mentioned are hereby incorporated by reference in their entirety.

Claims (29)

An optically clear biosynthetic implantable corneal device comprising crosslinked collagen, wherein the crosslinked collagen uses 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Implantable corneal device produced by the process of crosslinking the collagen polymer, comprising collagen in an amount of 5 to 50% (w / v). The corneal device of claim 1, wherein the amount of collagen is at least 6% (w / v). The corneal device of claim 2, wherein the amount of collagen is at least 10% (w / v). The corneal device of claim 3, wherein the amount of collagen is 10% (w / v) to 30% (w / v). The corneal device of claim 4, wherein the amount of collagen is 10% (w / v) to 24% (w / v). The corneal device of claim 1, wherein the crosslinked collagen contains one collagen. The corneal device of claim 1, wherein the crosslinked collagen contains two or more types of collagen. The corneal device of claim 1, further comprising a cell growth promoter. The corneal device of claim 8, wherein the cell growth promoter is a peptide. The corneal device of claim 9, wherein the peptide is a peptide having the amino acid sequence RGD, YIGSR or IKVAV. The corneal device of claim 8, wherein the cell growth promoter is selected from the group consisting of neurotrophic factor, nerve growth factor and epidermal growth factor. The corneal device of claim 11, wherein the cell growth promoter is distributed throughout the device. The corneal device of claim 1, wherein the collagen is the only water-expandable polymer of the device. The corneal device of claim 1, wherein the collagen is crosslinked at a pH below 6.0. 15. The corneal device of claim 14, wherein the pH is from 5.0 to 5.5. The corneal device of claim 1, wherein the crosslinked collagen comprises atelocollagen, type I collagen, type III collagen, or a combination thereof. The corneal device of claim 1, wherein the crosslinked collagen comprises recombinant collagen. The corneal device of claim 1, wherein the crosslinked collagen comprises prion free collagen, isolated from an animal. 19. The corneal device of claim 1, wherein the crosslinked collagen is produced by a process of crosslinking the collagen polymer using a nontoxic crosslinker, wherein the crosslinking is a zero length bond. The corneal device of claim 19, wherein the pH of the process is maintained to prevent pH surge. The corneal device of claim 1, further comprising poly (N-isopropylacrylamide-co-acrylic acid), chondroitin sulfate, N, O-carboxymethylchitosan, hyaluronic acid, hyaluronic acid aldehyde or alginate. The corneal device according to any one of claims 1 to 18 and 21, which is effective for the recovery of contact sensitivity after transplantation. combining the collagen polymer with a crosslinking agent at a pH below 6.0; And Curing the resulting blend to form a composition comprising crosslinked collagen 22. A method of manufacturing a corneal device of any one of claims 1 to 18 and 21 comprising a. The method of claim 23, wherein the blending step comprises mixing the collagen polymer and the crosslinking agent in a system configured to provide high shear forces on the resulting blend. The method of claim 23 wherein the blending is carried out at a temperature of from 0 to 5 ° C. 24. The method of claim 23, further comprising adding a cell growth promoter to the formulation. 22. The cornea of any of claims 1-18 and 21, wherein the corneal device is in contact with an eye of an individual having an ocular disease, disorder or injury for treating the ocular disease, disorder or injury. Device. 22. The corneal device of any one of claims 1-18 and 21 for treating said ophthalmic disease, disorder or injury in a subject in need thereof. The corneal device according to any one of claims 1 to 17, which exhibits a white light transmittance of at least 85%.

Images